One of the earliest events in the formation of a synapse between nerve and muscle--the neuromuscular junction--is the aggregation of acetylcholine receptors (AChRs) in the region of the muscle membrane contacted by nerve. This aggregation, or clustering, of AChRs involves both intracellular and extracellular macromolecules. Similar molecules participate in AChR clustering in vitro, in muscle cultured in the absence of nerve. The AChR clusters of cultured rat myotubes can now be purified greater than or equal t0 20-fold with little apparent change in organization. Such preparations are an excellent starting point for studying the macromolecules involved in maintaining AChR clusters. This grant proposes to use biochemical, immunological and fluorescence techniques to study this question. Rabbit antibodies to known cytoskeletal proteins, and monoclonal antibodies to purified AChR clusters, will be generated and used in immunofluorescence experiments to identify and localize macromolecules within AChR clusters. By preparing clusters in different ways, and by selectively extracting molecules from clusters, the localization of the antigenic sites, and their relative importance for maintaining AChR clusters should be learned. Such experiments have already shown that an actin-like molecule is important for AChR clustering. Using biochemical and immunological methods, this putative actin will be compared to actins purified from other sources. Using detergent solubilization, perhaps in conjunction with mild, reversible crosslinking, evidence for a complex of this putative actin with AChRs will be sought. If found, purified actin will be added to membrane fragments containing AChRs but stripped of endogenous actins, to learn, first, if it binds, and second, if its binding reduces the mobility of AChRs. For the latter, fluorescence photobleaching recovery will be used. The information obtained from these experiments will further be used to study the sequence of events involved in the assembly of the postsynaptic membrane. Fluorescent postsynaptic macromolecules will be injected into muscle about to synapse with nerve, and the distribution of the label, relative to AChRs, will be followed by time lapse video recording. Finally, the ability to separate clustered from nonclustered AChRs allows detailed comparison of their properties. The metabolic, pharmacological and biochemical properties of these two receptor populations will be compared. The information learned from the research outlined here should elucidate some of the early, and basic, events in the formation of the neuromuscular junction.